Receptor signaling pathways and intracellular signaling by receptor tyrosine kinases are intimately involved in cell growth and differentiation. The binding of a particular growth factor or cellular ligand to its receptor on a cell's plasma membrane can stimulate a wide variety of biochemical responses, including changes in ion fluxes, activation of various kinases, alteration of cell shape, transcription of various genes and modulation of enzymatic activities in cellular metabolism.
Many cell receptors are tyrosine kinases whose signaling is dependent upon tyrosine phosphorylation of both the receptor and other molecules. Specific phosphorylated tyrosine residues on these receptors recruit soluble intracellular signaling molecules to the receptor-ligand complex upon extracellular ligand stimulation, thus initiating the intracellular signaling cascade that involves secondary signal transducer molecules generated by the activated receptor. The signal can then proceed through a series of steps to the nucleus and other subcellular locations where the final effects of activation by the extracellular ligand are produced. Recruitment of other molecules in the signaling pathway is often accomplished by adapter molecules, which contain only protein-protein interaction domains (e.g., SH2 and SH3 domains) and have no associated enzymatic activity. By isolating and characterizing the adapter proteins and the molecules that interact with these adapters, important parts of the signaling mechanism can be discovered, monitored and controlled.
For example, one such adapter protein is Grb2, a 24–25 kDa cytosolic adapter protein containing two SH3 domains flanking an SH2 domain, which is known to be involved in linking many important molecules in receptor-ligand signal transduction (E. J. Lowenstein et all., 1992, Cell, 70:431–442 and J. Downward, 1994, FEBS Letters, 338:113–117). The central SH2 domain of Grb2 binds to an autophosphorylation site on the receptor and the two flanking SH3 domains link to intracellular effector target molecules. An example of one such target molecule is the mammalian homolog of the Drosophila ‘son of sevenless’ (SOS) protein, which is a guanine nucleotide exchange factor for ras; thus, Grb2 links receptors with the ras signal transduction pathway. It is now known that the SH3 domains also link to a number of other proteins involved in the signaling pathway, including Vav (R. Ren et al., 1994, Genes Dev., 8:783–795; J. Wu et al., 1996, Immunity, 4:593; and L. Tuosto et al., 1996, J. Exp. Med., 184:1161); c-abl (Z. S. Ye and D. Baltimore, 1994, Proc. Nat'l Acad. Sci. USA, 91:12629–12633); dynamin (I. Gout et al., 1993, Cell, 75:25–36); and SLP-76 (J. K. Jackman et al., 1995, J. Biol. Chem., 270:7029–7032). In addition, several other binding proteins have been noted during B- and T-cell signaling (See, e.g., K. Reif et al., 1994, J. Biol. Chem., 269:14081–14087 and D. G. Motto et al., 1994, J. Biol. Chem., 269:21608–21613).
The SLP-76 family of adapter protein molecules includes the SLP-76, BLNK and Clnk proteins (P. S. Myung et al., 2000, “Adapter proteins in lymphocyte antigen-receptor signaling”, Curr. Opin. Immunol., 12:256–266 and M. Y. Cao et al., 1999, “Clnk, a novel SLP-76-related adapter molecule expressed in cytokine-stimulated hemopoietic cells”, J. Exp. Med., 190:1527–1534). Expressed exclusively in cells of hematopoietic origin, these adapter proteins are involved in intracellular signal transduction. SLP-76 is an SH2 domain-containing 76 kDa leukocyte protein l:hat undergoes tyrosine phosphorylation following activation of the T-cell antigen receptor (TCR). SLP-76, upon tyrosine phosphorylation, interacts with Grb2 and phospholipase C-γ (PLC-γ), (J. K. Jackman et al., supra). The phosphorylation of SLP-76 on tyrosine is required for TCR-mediated cytokine secretion.
SH2 domain-containing proteins bind phosphorylated tyrosine residues and transmit important intracellular signals in many cell types. In the immune system, SH2 domain-containing proteins, such as SLP-76 and BLNK, play crucial roles in T-cell and B-cell activation. Therefore, SH2 domain-containing proteins are likely to be important targets for therapeutic intervention in immunological disorders, including autoimmune disorders and inflammatory indications. In addition, a partial sequence containing only a middle portion of a MIST/Clnk protein isolated by R. Goitsuka et al. (2000, Int. Immunol., 12:573–580) was implicated as being involved in receptor-mediated mast cell degranulation, thus providing another type of hematopoietic cell in which such SH2-domain-containing proteins function to transmit intracellular signals.
With particular regard to B-cells, cell function is dependent on the ability of the membrane B-cell receptor (BCR) to bind to antigen and induce an efficient cascade of intracellular biochemical signaling events from the membrane to the nucleus. These events culminate in the cytosol to rearrange the morphology of the cell through cytoskeletal reorganization and in the nucleus to activate the transcription of new genes to promote cellular proliferation and differentiation. Such biochemical and cellular mechanisms are required for B-cells to mature and function to produce an efficient immune response to foreign pathogens. Conversely, the abnormal activation of B-cells can lead to unregulated cellular proliferation and uncontrolled clonal expansion, resulting in B-cell tumors, lymphomas and leukemias. In addition, unregulated activation of B-cells may also contribute to a variety of autoimmune diseases mediated by self-reactive antibodies.
Similarly, in the case of T-cells, unregulated activation of the TCR can lead to aberrant T-cell growth, resulting in, for example, T-cell tumors, lymphomas, leukemias and thymomas. Thus, the ability to modulate TCR- and BCR-mediated signaling events may provide a rational approach to the treatment of T- and B-cell mediated tumors, and the like, as well as provide therapies for autoimmune diseases in which aberrant B-cell activation may be the culprit for cell destruction by auto-reactive antibodies.
Because aberrant or uncontrolled regulation of the cellular processes involved in cell growth can have disastrous effects, it is important to elucidate and gain control over these processes. This requires identifying molecules that participate in the signaling events that lead to mitogenesis and dissecting their functions and mechanisms of action. The identification of these participants is important for a wide range of diagnostic, therapeutic and screening applications. More specifically, by knowing the structure of a particular participant in a receptor ligand activation cascade, one can rationally design compounds that affect that cascade, to either activate an otherwise inactive pathway, or inactivate an overly active pathway.
Similarly, having identified a particular molecule in a ligand receptor cascade, situations in which that cascade is defective can also be identified and intervention can be achieved by means of therapeutic compounds or drugs, to prevent the development of a particular pathological state. The identification of participants in particular receptor ligand activation cascades and intracellular signaling events is thus of critical importance for screening compounds that affect these cascades and events, and treating a variety of disorders resulting from anomalies in these cascades and events as therapeutic agents. The present invention meets these and many other needs.
In addition, the discovery of human MIST, a new member of the SLP-76 family of adapter proteins, and the polynucleotide encoding this protein provides the art with new compositions and methods of use and treatment for the diagnosis, screening, monitoring, therapy, and prevention of immune system related conditions or diseases, particularly those involving T-cell and B-cell neoplasms, and inflammation disorders, diseases and conditions, particularly those involving hyperactivity of B-cells and T-cells, or other immune cells, such as mast cells or eosinophils.